Published ahead of print on January 7, 2004, doi:10.1164/rccm.200308-1167OC
© 2004 American Thoracic Society
Aggregations of Lymphoid Cells in the Airways of Nonsmokers, Smokers, and Subjects with AsthmaFaculty of Regional Professional Studies, Edith Cowan University, Bunbury; West Australian Sleep Disorders Research Institute, Sir Charles Gairdner Hospital; and School of Pharmacology and Medicine, University of Western Australia, Nedlands, Western Australia, Australia Correspondence and requests for reprints should be addressed to Alan L. James, West Australian Sleep Disorders Research Institute, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia. E-mail: ajames{at}it.net.au
Persistent airway inflammation is present in cases with asthma and in smokers with airflow obstruction. Isolated aggregations of lymphoid cells (IALC) may be sites of localized inflammatory cell activation. Their distribution and characteristics in cartilaginous airways were assessed in postmortem tissue from nonsmokers (n = 10), smokers (n = 9), and cases of nonfatal (n = 10) and fatal asthma (n = 10). IALC were present in 70–100% of cases, were more often in proximal than distal airways, and 80% were confined to the outer airway wall. IALC with area greater than 0.1 mm2 were more frequent in both asthma groups (p < 0.001). Airways with IALC had increased airway dimensions and greater numbers of eosinophils and lymphomononuclear cells. Within IALC, T and B lymphocytes were segregated and comprised more than 90% of all cells. Proliferating, apoptotic, and antigen-presenting cells (Rel B+ and HLA-DR+) were less than 5%, 30–40%, and less than 1% of all cells, respectively, and were similar in each case group. Vascular structures were increased (p < 0.01) in cases of fatal asthma. These findings show that, even in nonsmoking cases and cases without asthma, IALC are common, show cellular organization, and are associated with airway wall inflammation and remodeling. It remains to be determined if IALC contribute to or result from persistent airway inflammation in asthma.
Key Words: airway inflammation • remodeling • asthma The large mucosal surface of the bronchial tree is exposed constantly to inhaled antigens. The detection of these antigens is achieved through antigen-presenting cells, the most potent of which are dendritic cells (1), although other cells may also present antigen (2, 3). In the current view of immune surveillance, dendritic cells process antigen, mature, and migrate to the regional lymph nodes, where they activate T cells and B cells (4), which proliferate and traffic back to the airway (5). Once activated, T cells are capable of secreting proinflammatory cytokines and mediators, which contribute to the infiltration of inflammatory cells and acute inflammatory events (6). The inflammatory process includes inhibitory and repair mechanisms, designed to limit tissue damage. Chronic inflammation may result in the overexpression of growth factors, the proliferation of structural cells, and the deposition of abnormal proteins (7, 8). This process is believed to cause an overall thickening of the airway wall and forms the basis of airway wall remodeling (9). Regional lymph nodes form part of the secondary lymphoid tissue, which includes organized subepithelial aggregations of lymphocytes, such as Peyer's patches in the gut or bronchus-associated lymphoid tissue (BALT). BALT is usually described as lying adjacent to the airway lumen and is characterized by structural features (10), including specialized lymphoreticular epithelium and high endothelial venules (11). It has been suggested that BALT is not present in the healthy adult lung (12, 13), although it has been reported in the fetal lung (14), and up to the age of 20 years and may play a role in the ontogeny of active immunity (15, 16). Smaller, isolated aggregations of lymphoid cells (IALC) are also observed in the airway wall and may play a role in antigen uptake and cell-mediated immune responses. IALC may develop in response to repeated antigen exposure. They have been reported in the airways of smokers (17), patients with chronic respiratory infections (18), and elite skiers (19). Airway inflammation and remodeling are consistently seen in patients with asthma (9, 20–22) and in cigarette smokers (21, 23–25). In asthma, airway inflammation improves after treatment (26); however, it persists even during remission of symptoms (27, 28) and recurs when treatment is withdrawn (26). In patients with chronic obstructive pulmonary disease and chronic bronchitis, inflammation persists after cessation of smoking (29, 30). What causes airway inflammation to persist despite prolonged antiinflammatory treatment and the absence of an external trigger remains unknown. Possible mechanisms may include persistent exposure to inhaled antigen or in situ stimulation of immune cells. IALC in the airways of patients with asthma and in smokers may provide a mechanism for localized in situ T cell and B cell stimulation. This study was undertaken to assess the distribution of IALC in the airways, characterize the nature and function of the cells within IALC, and study the relation of IALC to airway dimensions and inflammation. Some of the results of theses studies have been previously reported in the form of an abstract (31).
Whole lungs were obtained from individuals coming to coroner's autopsy. This study was approved by the Sir Charles Gairdener Hospital's Ethics committee. Subjects were chosen where the cause of death was sudden, without chest trauma, and not due to respiratory causes (other than asthma) or other causes likely to affect the lung. Information regarding history of asthma, asthma symptoms, medication use, smoking and time from work, school or hospitalization due to asthma was obtained from next of kin, hospital files, coroner's files, and the subjects' medical practitioner. Cases were classified as nonsmokers without asthma (nonsmokers), smokers without asthma (smokers), nonfatal asthma cases, and fatal asthma cases. The left or right lung was fixed by inflation (20 cm H2O) in 20% buffered formalin for at least 24 hours. Blocks from consecutive generations of bronchi from the upper, middle, and lower lobes were embedded in paraffin. Sections (5 µm) were cut and stained with hematoxylin and eosin. The area of the inner and outer airway wall (32), airway smooth muscle, submucosal mucous gland, and cartilage were measured by direct tracing and expressed as area per millimeter of basement membrane. Measurements were undertaken with the observer blinded to the classification of each case. IALC were defined as focal collections of more than 50 lymphomononuclear cells with a cell density (cells/mm2) of more than 10 times that of the surrounding airway submucosa (Figure 1) . The number, area, and location of each IALC within each airway were measured. The percent of vascular structures within IALC was determined using point counts in random high-powered (x1,000) fields. Total eosinophil numbers were counted and lymphomononuclear cells were sampled (20) in the inner airway wall at high power (x400). Cells were not counted if they were in blood vessels, mucous glands, or mucous gland ducts.
From a subgroup of cases identified as having IALC, further sections (5 µm) were cut and stained using the immunoperoxidase technique (33) with monoclonal antibodies to identify cells as follows: macrophages (HAM56; Dako, Sydney, Australia), cells expressing major histocompatibility complex Class II (human leukocyte antigen-DR [HLA-DR]; Dako), B cells (CD20; Dako), proliferating cell nuclear antigen (PCNA; Dako), vascular endothelial cells (Factor VIII–related antigen; Dako), and antigen-presenting cells (Rel B; Santa Cruz, Melbourne, Australia). Double-labeled sections stained for cells undergoing apoptosis (terminal deoxynucleotidyl transferase–mediated dNTP nick end-labeling [TUNEL]; Roche, Indianapolis, IN) and for memory T cells (CD45RO; Dako, Glostrup, Denmark) were also prepared (34). The percent of positive cells within IALC was determined by counting a total of 200 cells in random high-powered (x1,000) fields.
The mean total area of IALC per airway was compared between case groups using a one-way analysis of variance, and the percentage of airways and cases containing IALC were compared between case groups using the
A total of 39 cases were examined: nonsmokers (n = 10), smokers (n = 9), nonfatal asthma cases (n = 10), and fatal asthma cases (n = 10). The causes of death, other than asthma, consisted of motor vehicle accidents (n = 10), suicide by hanging or self-poisoning (n = 13), sudden cardiac death (n = 4), ruptured cerebral aneurysm (n = 1), and liver disease (n = 1). There was no history of respiratory disease, respiratory symptoms, or other illness in the smokers and nonsmokers. All groups had similar distributions of sex, age, and time to fixation of the lung from death, with an overall mean of 27 ± 14 hours. We were unable to determine the time from death to fixation in one nonsmoker, one smoker, and three cases of nonfatal asthma. Four cases of nonfatal asthma and one case of fatal asthma were current smokers and two cases of fatal asthma were ex-smokers (Table 1) . The cases of nonfatal asthma were of mild clinical severity in that they had few symptoms and required mainly short-acting bronchodilators for treatment and had no time from work or school. In contrast, the cases of fatal asthma had frequent symptoms, reduced lung function due to asthma, and most required treatment with inhaled and oral corticosteroids. The duration of treatment in the asthma groups is unknown. Both large and small cartilaginous airways (n = 194) were examined. The mean (± SD) size of airways from each case group was similar with basement membrane perimeters of 18.5 ± 3.4, 19.9 ± 2.3, 19.6 ± 2.5, and 19.4 ± 3.6 mm for nonsmokers, smokers, nonfatal asthma cases, and fatal asthma cases, respectively.
Of all airways examined, 45% contained IALC and, of these, 41 airways (47%) contained more than one lymphoid aggregate. All case groups had subjects (70–100%) with IALC (Table 2) . There were no differences between the case groups with regard to the percent of cases or airways that had IALC or the percent of airways with multiple IALC. Of the 154 individual IALC observed, 80% were confined to the outer airway wall, in close proximity to submucosal mucous glands, mucous collecting tubules, or mucous ducts (Figure 1). Only 20% of IALC were confined to the inner airway wall between the basement membrane and the smooth muscle layer (Figure 2A) . This distribution was similar in all groups. There were no differences in the areas of IALC between lobes of the lung either within or between case groups; however, in smokers and in cases with asthma, the proximal airways had more IALC than the distal airways (Table 2). The frequency distributions of the cross-sectional area of individual IALC showed that approximately 12% of IALC were over 0.1 mm2 in area in the groups without asthma compared with more than 40% in both groups with asthma (p < 0.001, Table 2). The area of IALC in absolute terms and as a percent of airway wall area comprising IALC tended to be greater in cases of asthma and smokers, compared with nonsmokers, although these differences were not statistically significant (Table 2).
The dimensions of airways and the number of lymphomononuclear cells and eosinophils in airways with and without IALC are shown in Figures 3 and 4 . In all case groups, airways with IALC had greater areas of inner wall (p < 0.05 in nonsmokers and nonfatal and fatal asthma cases), outer wall (p < 0.05 in nonsmokers and nonfatal asthma cases), smooth muscle (p < 0.05 in smokers), submucosal mucous gland (p < 0.05 in nonfatal asthma cases, p < 0.01 in nonsmokers and smokers), and greater number of eosinophils (< 0.05 in nonsmokers and smokers, p < 0.01 in fatal asthma cases) and lymphomononuclear cells (p < 0.05 in fatal asthma cases, p < 0.01 in nonsmokers and nonfatal asthma cases) compared with airways without IALC. There were no differences between airways with and without IALC with regard to area of cartilage. In the group of nonsmokers, the airways with IALC had a longer basement membrane length ("bigger" airways) compared with airways without IALC (p = 0.045). However there were no differences in basement membrane length in airways with or without IALC in any other group.
Multiple regression analysis showed that the presence of IALC was independently associated with greater (p < 0.01) areas of inner and outer airway wall, airway smooth muscle, and submucosal mucous gland and a greater number of eosinophils (p < 0.01) and lymphomononuclear cells (p < 0.05). There was a significant inverse relationship (r = -0.33, p = 0.04) between age and the IALC area, and increasing age was also independently associated with increased areas of inner and outer airway wall, airway cartilage (p < 0.01), and submucosal mucous gland (p < 0.05). A history of asthma was associated with an increased area of airway smooth muscle, submucosal mucous gland, airway cartilage, and a greater number of eosinophils (p < 0.05). The IALC showed structural organization with regard to the distribution of lymphocytes. CD20+ cells (B cells) were rarely observed in the remainder of the airway wall, being confined almost exclusively to IALC where they comprised approximately 50–60% of all cells within IALC (Table 3) . CD20+ cells clustered together within IALC although the pattern varied between a central core of CD20+ cells, an outer ring of CD20+ cells, a separate half of CD20+ cells, and rarely a mixture of CD20+ and CD20- cells. These patterns were not systematically different between groups. On separate sections, CD45RO+ made up 40–80% of all cells. Airways containing IALC and labeled with CD45RO+ were not available for the smoking cases. Rel B+ or HLA-DR+ cells were less than 1% and 2–11% of cells in IALC, respectively. Proliferating cells (PCNA+) made up 2–5%, and cells undergoing apoptosis (TUNEL+) constituted 30–40% of cells. Macrophages comprised 1–2% of cells. Granulocytes (eosinophils and neutrophils) were rare. There were no differences between groups with regard to specific cell types. The percent area of IALC occupied by vascular structures was significantly greater in cases of fatal asthma (p < 0.01) compared with nonsmokers. All vascular structures (Figure 2B) were Factor VIII positive.
In this study we examined the distribution of IALC in the airways of nonsmokers and smokers without asthma and in cases of nonfatal and fatal asthma and found that from all groups, IALC were present in 45% of all airways examined, including 38% of all nonsmokers airways. Cases with asthma tended to have larger and greater numbers of IALC than cases without asthma, regardless of asthma severity; however, the distribution of IALC in the airways was otherwise similar in all case groups. IALC were distributed evenly in the upper, middle, and lower lobes and were more often present in the central than peripheral airways in all groups, except for the nonsmokers. The presence of IALC was associated with increased airway wall thickness, increased area of airway smooth muscle, and increased numbers of eosinophils and lymphomononuclear cells and was inversely related to age. IALC showed structural organization with separation of CD20+ and CD45RO+ cells. Cells likely to be presenting antigen comprised less than 10%, usually less than 2%, of total cell number. Lymphocytes in IALC showed low levels of proliferation, although apoptotic cells comprised 30–40% of all cells. There were no significant differences between smokers and nonsmokers and cases with and without asthma with regard to specific cell markers. To validate our definition of an IALC, we sampled the densities of cells in the 10 largest and 10 smallest IALC and compared these with the density of lymphomononuclear cells elsewhere in the airway wall. All IALC had a cell density (cells/mm2) that was 10 times that of the adjacent airway wall. We also counted the total number of cells in the 10 smallest IALC with such a cell density and found that only one lymphoid aggregate had less than 100 cells. Therefore, we concluded that the definition for IALC as being a focal collection of more than 50 lymphomononuclear cells, with a density greater than 10 times that of the surrounding airway wall, reliably distinguished these aggregations of cells. We did not use an unbiased stereologic approach to sampling IALC in airway sections, so it is difficult to comment on the true volume of IALC and variations in their cellular organization (see below) in three dimensions. In addition, it is likely that had we taken more sections from each case, we would have approached 100% of cases having IALC in control airways. However, given that sections were taken randomly, it is likely that larger or more frequently occurring IALC would be included and available for measurement. The IALC we have described in this study did not have the same structural features as BALT described by Sminia and coworkers (10) but were present in 70% of nonsmokers and 80% or more of smoking control cases or cases of asthma. It has been suggested that BALT is not present in normal healthy human lung tissue but that its formation is a result of inflammation (12). Delventhal and coworkers (35) stimulated the formation of BALT in pigs, a species in which BALT is not frequently found, by exposing them to bacteria. In further work, the same group reported that only 8% of patients with chronic bronchitis and bronchiectasis had BALT (36). Richmond and coworkers (17) observed BALT in 82% of smokers but only in 14% of nonsmokers. They defined BALT as aggregations of mature lymphoid cells closely related to the bronchial epithelium of the airway lumen or bronchial gland ducts, although in both their study (17) and in that of Bosken and coworkers (37) in normal subjects and those with airway obstruction, "BALT" was predominantly seen in the outer airway wall, as in the present study. We also observed that proximal airways had more IALC than distal airways in the smoking control cases and both asthma groups but not in the nonsmoking control cases. This may reflect greater proximal deposition of inhaled antigens and particles in cigarette smoke in the conducting airways. The findings from the present study and from other studies suggest that IALC, rather than BALT with specialized epithelium, are an integral feature of the normal proximal airway. IALC have been reported in the airways of patients with cystic fibrosis and have been reported to be increased in size compared with donors at the time of transplantation (38). Sue-Chu and coworkers (19) reported IALC in bronchial biopsies at the second and third generation carinae in the lungs of elite cross-country skiers with asthma-like symptoms. In that study, 64% of skiers were reported to have IALC compared with 25% of nonsmoking control cases. Sue-Chu and coworkers (19) defined aggregates as more than 50 cells, as we did; however, their lower prevalence of IALC is not unexpected because the airways were sampled using small bronchial biopsies, which will mainly sample the inner airway wall. We observed that only 20% of IALC from nonsmokers were in the inner airway wall. The observations from these studies suggest that chronic inflammation (19) or infection (38) may be associated with the development of IALC. This is supported by observations in a mouse model of allergic airway inflammation (39). In the present study, the airways that contained IALC had thicker walls, more smooth muscle and submucosal mucous gland and had greater numbers of eosinophils and lymphomononuclear cells, compared with airways without IALC. These findings are consistent with the hypothesis that the presence of IALC in the airway wall may contribute to persistent inflammation and airway wall remodeling. Alternatively, IALC may simply develop in sites of the airway that are subject to greater inflammatory stimulation. The current paradigm of surveillance in adaptive immunity is that migratory antigen-presenting cells, the dendritic cells, capture and process antigen and traffic to regional lymph nodes where antigen is presented, together with appropriate costimulatory molecules, to B cells and T cells that then become activated. T cells then traffic back to site of antigen and coordinate the inflammatory response. Much of this process is controlled by chemokine receptors expressed on inflammatory cells and their ligands, expressed on lymph vessels, high endothelial vessels, and within the highly organized zones of the lymph node (40). Recent work however has shown that antigen-presenting cells do not have to migrate into secondary lymph tissue to successfully prime T cells (41). Localized priming or activation of T cells and B cells could take place in IALC within the airway wall. The IALC observed in the present study showed considerable structural organization with separation of CD20+ B cells and CD45RO+ T cells. There was some overlap in the percentage of the two cell types in same groups, particularly in the fatal cases of asthma. Circulating B cells may express CD45RO in some disease states (42), and given the increased vascular structures in the fatal asthma group, it is interesting to speculate that increased trafficking of B cells into IALC may occur in fatal asthma.
Macrophages identified morphometriclly or as HAM56+, comprising up to 2% of cells and granulocytes (eosinophil and neutrophils), were rare. Currently there are no specific methods of identifying dendritic cells in formalin-fixed tissues. Therefore, to investigate antigen presentation, we labeled cells with antibody to human leukocyte antigen-DR that identifies major histocompatibility complex Class II molecules expressed on the cell surface and that are necessary for successful antigen presentation (43). In addition, we identified expression of nuclear Rel B. Translocation of Rel B, a component of the proinflammatory transcription factor nuclear factor- Trafficking of memory T cells to peripheral tissues and secondary lymphoid tissue is controlled by chemokine receptors (such as CCR7) on the T cell and their specific ligands (such as secondary lymphoid tissue chemokines) expressed on high endothelial venules (45, 46). In addition, inflammatory cells, especially specific memory T cells, may be captured by the expression of integrins and selectins on surface of high endothelial venules (47). We observed numerous vascular structures within IALC, which were increased in area in fatal asthma, compared with nonsmoking cases without asthma. Vascular structures were observed frequently in the study of Richmond and coworkers (17) although as in the present study, specific markers for high endothelial venules and endothelial chemokine receptor ligands and integrins were not reported. Further characterization of the vascular structures seen in IALC in airways is required. The IALC we observed appeared to differ from both BALT and lymph nodes. Unlike BALT, the IALC had no specialized epithelium and they were mostly seen away from the luminal epithelial surface. Although close to mucous glands, there was no consistent association or modification of the glandular epithelium, and apart from sloughed areas of epithelium the mucous gland ducts were devoid of inflammatory cells. Unlike lymph nodes, the IALC lacked any evidence of a capsule or apparent germinal centers. Lymph nodes also contain efferent lymphatic vessels, which lack Factor VIII staining (48). We did not identify any Factor VIII–negative vascular structures within IALC. The mucosal surface of the airways need continuous surveillance to detect and respond to potentially harmful antigens. IALC may play a part in this surveillance by way of localized antigen uptake and initiation of immune responses. Our findings challenge the notion that aggregations of lymphocytes in the airway wall are not an integral part of the normal healthy lung. The airway submucosa of smokers with chronic obstructive pulmonary disease and patients with asthma show persistent and localized inflammation. Whether IALC contribute to this inflammation or whether they result from persistent inflammation under the influence of cytokines such as a tumor necrosis factor or lymphotoxin (49) remains unclear; however, their role in mucosal defense of the airways warrants further clarification.
The authors thank Prof. Maurizio Vignola and Ms. Liboria Siena (Palermo) for preparing the CD45RO and terminal deoxynucleotidyl transferase–mediated dNTP nick end-labeling.
Supported by the National Health & Medical Research Council of Australia and a grant from the Western Australian Institute for Medical Research. Conflict of Interest Statement: J.G.E. has no declared conflict of interest; C.M.J. has no declared conflict of interest; S.M. has no declared conflict of interest; J.P.L. has no declared conflict of interest; N.G.C. has no declared conflict of interest; A.L.J. has no declared conflict of interest. Received in original form August 21, 2003; accepted in final form January 6, 2004
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ABSTRACT
METHODS
2 test. Airway dimensions and the density of cells were compared between airways with and without IALC using the Student's t test. The independent effects of IALC area, history of asthma, smoking, sex, and age on airway dimensions and cell density were examined using multiple linear regression analysis. Statistical analysis was undertaken using SigmaStat for Windows version 2.03 (SPSS Inc., Sydney, Australia). 
B, is necessary for the expression of CD40 on antigen-presenting cells (
